Linear furocoumarins, also known as psoralens, have been used in combination with ultraviolet light for centuries in cosmetics and for the treatment of proliferative skin diseases such as, for example, vitiligo, eczema, mycosis fungoides, and psoriasis. Terms such as photosensitization, photochemotherapy, photopheresis and PUVA (psoralens ultra violet A radiation) are commonly used to refer to such methods. Recently it was discovered that by modifying the administration of psoralen and ultraviolet light to an offending condition, psoralens can be used to treat cancer (e.g., T cell lymphoma), autoimmune diseases, and microbial infection.
The basic structure of psoralen, with the ring numbering structure used throughout the specification, is shown below: ##STR2##
All psoralens contain two photo-activatable functions (absorbing in the UVA range)--an aryl-conjugated unsaturated pyrone (the coumarin portion) and an aryl-conjugated vinyl ether (the furan portion). All of the commercially available psoralens are highly lipophilic, non-nitrogenous, uncharged small molecules with minimal water solubility. Commercial psoralens are used in over-the-counter cosmetic creams, prescription pharmaceuticals, and as investigational candidates for many of the uses described above. The commercial psoralens in cosmetic/medical use include methoxsalen (also known as xanthotoxin, 8-methoxypsoralen or 8-MOP), trisoralen (also called 4,5',8-trimethylpsoralen, TMP, or trioxsalen), and bergaptan (alternatively named 5-methoxypsoralen or 5-MOP).
The phototherapeutic action of psoralens has been discussed for example, by J. E. Hearst, "Photochemistry of the Psoralens," Chemical Research in Toxicology, 2, 69, (1989)) and T. F. Anderson and J. J. Vorhees, Annual Reviews of Pharmacol. and Toxicol., vol. 10, p. 177, (1982). According to these articles, the highly lipophilic psoralens penetrate the target cell's membrane, intercalate into nuclear DNA, and photo crosslink the double helix through bis-cyclobutanes generated from the 3,4-double bond and the 4',5'-double bond [see numbering shown above] to double bonds in DNA's pyrimidine bases. Thus, because the crosslinked DNA is unable to uncoil and function as a template for new gene expression, the target cell is rendered non-viable.
A severe limitation to the acceptance of psoralen-based photochemotherapy or cosmetic skin pigment enhancement, however, is the risk of genetic mutations induced by DNA damage since the natural cellular level repair processes of bi-functional DNA-crosslinks are highly error-prone. Errors in cellular repair processes of true crosslinks translate to mutagenic/carcinogenic events and, in the clinical use of psoralens, represent a significant post-treatment risk of cancer. See, for example, R. S. Stern et al, "Cutaneous Squamous-cell Carcinoma in patients treated with PUVA," New England J. of Med., pp. 1156-116 (1984); R. S. Stern et al, "Malignant Melanoma in Patients Treated for Psoriasis with Methoxsalen and Ultraviolet A Radiation (PUVA)," New England J. of Med., vol. 336, pp 1041-1045 (1997); and W. L. Morrison et al. "Consensus Workshop of the Toxic Effects of Long-Term PUVA Therapy," Arch. Dermatol., vol. 134, pp. 595-598 (1998).
The use of nonlinear furocoumarins (known as angelicins) for the treatment of psoriasis and other skin diseases is taught, for example by U.S. Pat. No. 4,312,883. According to the patent, nonlinear furocoumarins are effective photochemotherapeutic compounds that do not have the risks associated with psoralens. Nonlinear furocoumarins, however, are limited by their structural geometry, forming only non-crosslinked monoadducts which have diminished mutagenic behavior. See, for example, R. S. Cole, "Repair of DNA Containing Interstrand Crosslinks in E. Coli," Proc. Nat. Acad. Sci., volume 70, p. 1064 (1973). Further, lipophilic linear psoralens, capable of forming only monoadducts, can be phototoxic to malignant cells. See J. VanDongen, N. D. Heindel et al., "Synthesis of Psoralen Analogs and Evaluation of their Inhibition of Epidermal Growth Factor Binding," J. Pharm. Sci., volume 80, No. 7, pp. 686-689 (July 1991).
Despite such risks, an alternative mechanism exists, not involving DNA, by which psoralens can act as phototoxins to a cell. A 22 kDa receptor protein present on psoralen-sensitive cells has been identified as a binding site for photo-activated psoralens. Binding a psoralen to this non-nuclear receptor followed by UVA light activation of the psoralen blocks subsequent binding of epidermal growth factor (EGF) to that receptor. The existence of this non-nuclear target has been described in J. D. Laskin et al., "A Possible Mechanism of Psoralen Phototoxicity Not Involving Direct Interaction with DNA," Proc. Nat. Acad. Sci., vol. 82, pp. 6158-6161, (September 1985).
U.S. Pat. Nos. 5,473,083 and 5,216,176 report that reduced and quaternized psoralens are valuable photo-activated therapeutics. Although promising as therapeutics, the 5'-subsituted dihydro quaternary compounds have often been extremely difficult to synthesize. See, for example, copending U.S. patent application Ser. No. 09/199,552, filed Nov. 25, 1998, the disclosure of which is hereby incorporated by reference. Furthermore, no previous method existed for synthesis of 4'-(N-pyridiniummethyl)-4',5'-dihydropsoralen.